Explore The Mechanism Of Autophagy In Browning Of 3T3-L1 Cells

Excessive proliferation and hypertrophy of white adipocytes of mammals can lead to obesity and its related metabolic diseases.Converting white adipocytes which store energy into beige adipocytes which produce heat by white adipocytes browning is one of the strategies to prevent obesity.Previous studies in our laboratory have shown that inhibition of insulin signaling by PI3K-AKT can not only inhibit the lipogenic differentiation of white preadipocytes and promote the dedifferentiation of white mature cells,but also significantly inhibit the human and mice preadipocytes browning.In addition,domestic and foreign studies have found that autophagy plays an important regulatory role in the process of lipogenic differentiation and dedifferentiation of white preadipocytes,and the key junction point of autophagy and lipogenic differentiation is mTOR.So,what role does autophagy play in regulating the Browning of white preadipocytes? We hypothesized that autophagy is involved in and regulates Browning of white preadipocytes through the PI3K-AKT-mTOR signaling pathway.To test this hypothesis,we designed the following experiments to preliminarily clarify the molecular mechanism of autophagy regulating Browning of white preadipocytes.In this study,3T3-L1 mouse fibroblast cell lines were selected as experimental materials and browning adipocyte differentiation was induced by "brown cocktail method" for a total of 8 days.During this period,7 groups were set up through different drug interventions: Simple induction group which was only added complete browning induction medium(CBIM),insulin signal suppression group(CBIM+OSI-906),not induction group which was only added complete growth medium(CGM),autophagy promoter group(CBIM+Rapa),autophagy inhibition group(CBIM+Baf-A1),insulin inhibits autophagy promoter group(CBIM+ OSI-906+Rapa)group and insulin inhibited autophagy inhibited group(CBIM+ OSI-906+Baf-A1).Induction was started from BD0 and continued to be induced to BD8.This article mainly through molecular biology detection means,in the transcription and translation level brown detection of the related molecules,a concomitant key molecular biomarkers and insulin signaling pathway,autophagy the change of the key molecules,and combining with morphological results,preliminary discussion autophagy,insulin signal,beige adipocyte differentiation into three interaction mechanism,test results are as follows.1.The insulin signal of 3T3-L1 preadipocytes was continuously inhibited(with the addition of OSI-906),and its morphology showed that: the lipid composition rate of insulin inhibition was much lower than that of the group only induced(non-insulin inhibition),and the lipid formation process was slower,but some adipogenic cells still existed.Transcription and translation level detection results showed that The expression levels of key insulin signaling molecules AKT,Browning marker molecules UCP1,PGC1?,lipid-related molecules PPAR? and C/EBP? were significantly inhibited,but the expression levels remained 20.55±2.35% compared with the uninduced group,while the expression levels of key molecules AMPK and SIRT1 in another Browning signaling pathway were promoted.This indicates that inhibition of the insulin receptor PI3K-AKT signaling pathway reduces Browning levels and lipid formation,and compensatively maintains certain Browning levels through the AMPK-SIRT1 signaling pathway.This is consistent with the results of previous studies in our laboratory,demonstrating that insulin signaling mainly regulates the Browning of adipocytes through the PI3K-AKT signaling pathway.2.During the induction process of 3T3-L1 beige adipocyte differentation,autophagy was continuously promoted or inhibited,and its morphology showed that after promoting autophagy,the process of beige adipocyte differentation was delayed and the lipomorphism rate was significantly reduced.On the contrary,inhibiting autophagy could accelerate the beige adipocyte differentation process while increasing the lipomorphism rate.Transcription and translation level test results showed that autophagy promoter group could significantly promote the expression of autophagy related genes(LC3,Beclin1)and SIRT1,while inhibit the expression level of mTOR,Browning related genes and lipid-forming molecules.The autophagy inhibition group significantly promoted the expression of mTOR,Browning related genes(UCP1,PGC1?)and lipid-forming molecules(PPAR?,C/EBP?),while the expression level of autophagy related genes was not significantly different from that of the induction group.The above results showed that promoting autophagy inhibited the expression of mTOR downstream of insulin signal,which inhibited insulin signal through feedback regulation to inhibit the Browning into lipid differentiation,on the contrary,inhibiting autophagy promoted the expression of mTOR,thus promoting Browning level and lipid formation process.3.In the inhibition of 3T3-L1 insulin signal,promote and inhibit autophagy intervention 3 t3-L1 into brown fat differentiation,its shape shows: compared with the insulin signal suppression group,insulin inhibit autophagy promotes group has fat rate lower,almost no fat,insulin and inhibit autophagy promotes group,its fat rate increased,and quickened the pace into fat;Transcription and translation level detection results showed that insulin signal suppression significantly inhibit the expression of mTOR and AKT,and autophagy marker genes(LC3,Beclin1)expression level rise significantly,compared with the insulin signal suppression group,insulin inhibit autophagy promotes group significantly promote autophagy biomarkers(LC3,Beclin1)expression of mTOR,AKT,brown related gene(UCP1,PGC1?)and a concomitant related molecules(PPAR?,C/EBP?)expression levels were significantly lower,and AMPK,SIRT1 expression get promotion;Different from the insulin-inhibited autophagy promoter group,there was no significant difference in the expression levels of autophagy marker molecules(LC3,Beclin1)in the insulin-inhibited autophagy inhibitor group,but the expression levels of mTOR,AKT,brown-related molecules(UCP1,PGC1?)and lipid-making molecules(PPAR?,C/EBP?)were significantly up-regulated.This indicated that inhibition of insulin signal promoted autophagy by inhibiting the PI3K-AKT-mTOR signaling pathway,and inhibition of Browning lipid formation and Browning level through mTOR feedback regulation.After inhibiting insulin signaling,autophagy is promoted,and mTOR is further inhibited by promoting the expression of AMPK,thereby further inhibiting Browning and maintaining Browning at an extremely low level through the AMPK-SIRT1 signaling pathway,which is almost fat-free in morphology.Similarly,inhibition of autophagy after inhibition of insulin signal will reduce the expression of AMPK,and then the expression level of mTOR will be upregned,and the inhibition of insulin signal will be weakened,thus increasing the Browning level to a certain extent.Combined with the above results,autophagy plays a key regulatory role in the process of browning adipocyte differentiation of 3T3-L1 through insulin mediated PI3K-AKT-mTOR signaling pathway,and its possible molecular mechanism is as follows: The important connection point between autophagy and insulin signal is mTORC1.When it is inhibited,it will activate autophagy on the one hand,and after activating autophagy,it will promote the expression of AMPK through ULK1,further inhibit mTORC1,and compensatively maintain browning level through the AMPK-SIRT1 signaling pathway.On the other hand,phosphorylation of AKT is inhibited by direct inhibition of PGC1? secretion or by feedback regulation of PI3K-AKT-mTOR signaling pathway,so that PGC1?,PPAR?,C/EBP? and PRDM16 can't form a transcription complex,thereby inhibiting the expression of UCP1 and thus inhibiting browning.